This proposal aims to elucidate how proanthocyanidin-rich extracts of grape polyphenols (GPs) modulate bile acid (BA) signaling to farnesoid X-receptor (FXR) resulting in improved glucose metabolism. Using intestine- specific (Fxr?IE) and liver-specific (Fxr?L) FXR knockout mice and derived ileal organoids, we will investigate whether GP-induced glycemic improvements are due to inhibition of intestinal and/or hepatic FXR signaling. We will also determine if GPs directly sequester secondary bile acids (SBAs) or indirectly leads to their depletion. Consumption of polyphenol-rich foods is associated with reduced risk of chronic disease; however, many dietary polyphenols are poorly absorbed raising questions about their mechanism(s) of action. Metabolic benefits of polyphenols are likely driven by changes in the gut microbiota. Prior work showed that GPs improved glucose metabolism in high-fat diet (HFD)-fed wild type (WT) mice due to GP-induced changes in the gut microbiota that correlated with attenuated body weight, metabolic endotoxemia (i.e. lower serum lipopolysaccharide (LPS)), tissue inflammation, as well as increased tight junction protein expression. Our recent data showed that GP supplementation mitigated hyperglycemia in diabetic db/db mice independent of changes in obesity, gut barrier integrity, and inflammation. Rather, GP-induced changes in the gut microbiota of db/db mice promoted a BA profile that suppressed intestinal FXR activity and FXR responsive pathways. Selective inhibition of intestinal FXR has been correlated with improved glucose metabolism and decreased ceramide synthesis in mice treated with antidiabetic drug, metformin. Metformin altered the gut microbiota and BA pool, reduced ceramide production, and lowered expression of hepatic gluconeogenic markers [Sun et al., 2018]. Dual antagonism of FXR in liver and intestine may promote cholestasis [Kong et al., 2012], therefore differentiating tissue specific effects of GPs on FXR activity is important. 16S rRNA V4 amplicon sequencing showed that GP supplementation depleted genera implicated in secondary BA (SBAs) metabolism concomitant with a dramatic decrease in serum SBAs and an increase in primary BAs (PBAs). Intestinal and liver tissues of mice supplemented with GPs had: 1) decreased gene expression of Shp and Fgf15, markers of FXR activity; 2) lowered expression of Smpd3, Cers4, and Sptlc2 , FXR responsive genes critical for ceramide synthesis; and 3) increased expression of Cyp7a1, an enzyme responsible for PBA synthesis. Gut organoids were used to differentiate the direct effects of BAs on intestinal FXR activation and ceramide synthesis and revealed GP-induced a BA profile antagonistic to intestinal FXR. We propose to 1) investigate glucoregulatory effect of GP supplementation in Fxr?IE and Fxr?L to uncover if GPs are antagonistic to liver FXR signaling mechanisms and distinguish the ability of GPs to sequester SBAs agonistic to FXR within the ileum. 2) Use ileal gut organoids derived from FXR?IE to determine the requirement of intestinal FXR for GP-mediated regulation of ceramide biosynthesis and 3) directly determine whether anaerobic cultures of ileal content collected from GP supplemented mice lack ability to produce SBAs.